For centuries, various natural and unnatural compositions and/or compounds have been added to foods, beverages, and/or comestible (edible) compositions to improve their taste. It has long been known that there are only a few basic types of “tastes” (sweet, sour, bitter, salty, and “umami”/savory). Sour and salty tastes are mediated by channel-type receptors. Sweet, bitter and umami tastes are mediated by G-protein coupled receptors (GPCRs) and second-messenger signaling cascades (Iwata et al., “Taste Transductions in Taste Receptor Cells: Basic Tastes and Moreover,” Curr. Pharm. Des. (Id.) (2013)).
One of the basic taste modalities that humans can recognize is bitter. The physiology of bitter and other taste modalities has become much better understood over the last decade, it is now known that bitter compounds elicit bitter taste by interacting with a family of cell surface receptors which belong to the superfamily of seven transmembrane domain receptors that interact with intracellular G proteins. Particularly, it is known that bitter ligands interact with one or more members of a family of GPCRs generally referred to in the art as T2Rs or TAS2R's.
The T2Rs are expressed in humans, rodents and other mammals (Adler et al., “A Novel Family of Mammalian Taste Receptors,” Cell 100(6):693-702 (2000); Chandrashekar et al., “T2Rs Function as Bitter Taste Receptors,” Cell 100(6):703-711 (2000); Matsunami et al., “A family of Candidate Taste Receptors in Human and Mouse,” Nature 404(6778):601-4 (2000)).
Human and other mammalian T2R genes are specifically expressed in subset of taste receptor cells of the tongue and palate epithelia. T2Rs are activated by gustducin, a G protein specifically expressed in taste cells and linked to bitter stimuli transduction (Wong et al., “Transduction of Bitter and Sweet Taste by Gustducin,” Nature 381:796-800 (1996)). Gustducin activation by mT2R5 occurs only in response to cycloheximide (Chandrashekar et al., “T2Rs Function as Bitter Taste Receptors,” Cell 100(6): 703-711 (2000)). The amino acid and nucleic acid sequences of hT2Rs have been previously reported and are disclosed in published PCT applications by Zuker et al. (WO 01/18050 A2, (2001)), U.S. Pat. No. 7,105,650 by Adler et al. and (WO 01/77676 A1 (2001) by Adler et al.) as well as in Senomyx U.S. Pat. Nos. 8,524,464; 8,445,692; 8,338,115; 8,318,447; 8,273,542; 8,221,987; 8,153,386; 8,076,491; 8,071,320; 8,030,468; 8,030,451; 8,030,009; 8,030,008; 8,017,751; 7,968,693; 7,939,671; 7,939,276; 7,932,058; 7,927,823; 7,915,003; 7,883,856; 7,816,093; 7,811,788; 7,794,959; 7,785,802; 7,776,561; 7,736,862; 7,723,481; 7,723,051; 7,718,383; 7,704,698; 7,638,289; 7,517,972; 7,407,765; 7,399,601; 7,396,651; 7,393,654; and 7,022,488; all of which are incorporated by reference in their entirety herein.
To date, 23 human T2R genes are known to be functional and have been deorphaned by various groups including the present assignee Senomyx Inc. High throughput screening methods have been used to identify compounds that activate or modulate, preferably block, bitter taste elicited by the interaction of specific bitter ligands and hT2Rs. hT2R blockers are useful as potential additives for incorporation in various foods, beverages, nutraceuticals, medicaments and other comestibles.
For example, in U.S. application Ser. No. 10/191,058 incorporated by reference herein in its entirety, the present assignee used high throughput screening assays to discover bitter ligands that specifically activate different human T2Rs. Additionally, in U.S. application Ser. No. 11/455,693, incorporated by reference herein, the present assignee further identified bitter ligands that specifically activate other human T2Rs.
Also, in International Application Publication No. WO 2011/106114 to Karanewsky et al., assigned to Senomyx, Applicants described the identification and synthesis of many bitter antagonist compounds. Further this PCT application disclosed a means for synthesis of substituted 1-benzyl-3-(1-(isoxazol-4-ylmethyl)-1H-pyrazol-4-yl)imidazolidine-2,4-diones by the synthetic scheme depicted schematically in FIG. 1 (WO 2011/106114) (“'114 PCT Application”). The synthetic procedure disclosed in the '114 PCT Application differs from the synthetic process which is in part the focus of this application. This previous process included a number of steps and in particular included a step resulting in the formation of N-benzylglycine ester 4′a; N-alkylation of a 4-carboxypyrazole 5′a; in situ preparation of isocyanate 10′a via a Curtius rearrangement; quenching of the intermediate 10′a with ester 4′a and cyclization to form the hydantoin moiety (11′a), and finally deprotection of the phenolic silyl ether and recrystallization from ethanol resulting in the formation of the compound referred to in the FIG. 1 as 12′a.
While this synthetic procedure is useful, it has features which may be problematic, especially insofar as its usage for large scale synthesis. For example, this synthesis procedure involves the use of hazardous hydrazine. In addition, the process includes a Curtius rearrangement which potentially presents safety problems associated with the uncontrolled release of nitrogen (Lieber et al., “Carbamoyl Azides,” Chem. Rev., 65(3):377-384 (1965); Anon., Sichere Chemiarb., 1984, 36, 143-144). Therefore, in part based on these disadvantageous features, there is a need for improved methods for the synthesis of substituted 1-benzyl-3-(1-(isoxazol-4-ylmethyl)-1H-pyrazol-4-yl)imidazolidine-2,4-diones, especially methods that are suitable for large scale synthesis of these compounds. The present invention achieves these objectives.